Recombinant attenuated Salmonella vaccines (RASVs) are an effective and inexpensive way to elicit strong mucosal and humoral immune responses. However, one problematic issue in this field has been that while RASVs are attenuated and safe in animal models, when administered to humans they can be reactogenic, producing unwanted side effects, including diarrhea, abdominal pain, gastrointestinal disorder, nervous system disorders, and fever. Many of these symptoms are consistent with known reactions to lipid A. Lipid A interacts with the human immune system via the toll-like receptor 4 (TLR4) which recognizes specific moieties associated with lipid A, including the acyl chains. The length and number of acyl chains influences the strength and outcome of the lipid A: TLR4 interaction. In an effort to reduce the potential for endotoxic effects in live bacterial vaccines,we will investigate the effects of modifying the acyl chain length in living Salmonella cells. Our goa is to reduce the overall toxicity of lipid A without compromising overall immunogenicity by modifying the length of the acyl chains from lipid A. We will construct a series of Salmonella mutants that differ in lipid A acyl chain length, and evaluate and compare their virulence and ability to elicit innate immunity in wild-type background. Acylation modifications that do not affet virulence or colonization ability will then be evaluated in Salmonella vaccines, to determine their influence on ability to elicit an immune response against a vectored antigen and against challenge. The success of this project could lead to safer and more immunogenic strains for human use. PUBLIC HEALTH RELEVANCE: Effective live recombinant attenuated Salmonella vaccines expressing heterologous antigen genes hold the promise of providing low cost, orally administered, life-long protection against a variety of diseases. The goal of this project is to enhance safety and immunogenicity by reducing the inherent toxicity of the lipid A component of Salmonella without compromising its adjuvant properties.